In response to infection, numerous cell types within the vertebrate immune system act in concert to effect the rapid and efficient clearance of the invading pathogen. Among these cell types are T cells, which develop in the thymus and which are responsible for cell-mediated immunity. T cells are divided into several major subclasses, including cytotoxic T cells, which kill virus-infected cells, as well as two classes of regulatory cells, called helper T cells (Th cells) and suppressor T cells, which act to modulate the activity of other immune cells. During chronic infections, Th cells develop into at least two phenotypically and functionally distinct effector populations, Th1 and Th2 lymphocytes. Th1 cells produce IFN-.gamma and IL-2, which are commonly associated with cell-mediated immune responses against various intracellular pathogens, whereas Th2 cells produce cytokines such as IL-4, IL-5, IL-6, IL-10 and IL-13, that are crucial to control extracellular helminthic infections.
In certain cases, the number, activity, or other properties of Th1 or Th2 cells can become abnormal, and these cell types can play a role in one or another disease or condition. For example, Th1 cells have been associated with organ-specific autoimmune diseases, delayed-type hypersensitivity, and transplant rejection. In addition, imbalance of Th2 cytokines are observed in various atopic and allergic diseases, which are usually accompanied by increased production of IgG1 and IgE as well as the activation of eosinophils and mast cells.
Cytokines such as IL-12 and IL-4 have dominant roles in determining the outcome of Th differentiation into Th1 and Th2 subsets, respectively. These cytokines bind to their cognate receptors, leading to activation of the Janus family of kinases and the latent transcription factors known as signal transducers and activators of transcription (STATs). For example, in Th1 cells, following the binding of IL-12 to its cognate receptor, STAT4 is activated, thereby leading to the production of IFN-.gamma. Accordingly, STAT4-deficient mice are defective in Th1 differentiation and do not respond to intracellular pathogens such as Listeria monocytogenes. In Th2 cells, IL-4 leads to the activation of STAT6, which is essential for the development of these cells. Accordingly, STAT6-deficient mice have an impaired ability to produce IL-4-secreting Th2 cells, thereby resulting in a failure to expel intestinal helminths. Interestingly, these STAT6 mutant mice are protected from antigen-induced airway hyperresponsiveness.
Additional reports have identified various genes that are differentially expressed in Th1 and Th2 cells. For example, the transcription factor ERM is selectively expressed in Th1 cells, and, in Th2 cells, GATA-3 and c-Maf are selectively expressed. GATA-3 is required for the expression of certain Th2 specific genes, can lead to the expression of IL-4 and IL-5 in Th1 cells, and inhibits the production of IFN-γ in Th1 cells. See, e.g., Zheng et al. Cell 89(4):587 (1997); Zhang et al., J. Biol. Chem. 272:21597 (1997); or Ferber et al., Clin. Immunol. 91:134 (1999). Additionally, several cell surface proteins are also differentially expressed in the Th1 and Th2 subsets. For example, Th1 cells express various chemokine receptors such as CXCR3, CCR1, and CCR5. Th2 cells, in contrast, express CD30 as well as various chemokine receptors such as CCR8.
Various cell surface proteins have been identified as having four-transmembrane domains, and are called tetraspanins, or transmembrane 4 superfamily (TM4SF). Such proteins, including, for example, CD4, CD81, CD9, and CD20, have a strong propensity to form molecular associations with other cell surface molecules. CD81, for example, which is expressed in both T and B lymphocytes, is found in a multimolecular complex with CD19 and the complement receptors 1 and 2 in B lymphocytes. Previous studies have demonstrated that this complex collectively regulates the threshold for antigen receptor-mediated B cell activation. In T cells, CD81 contributes to cell proliferation as well as to IL-2 and IL-4 production. Other four transmembrane proteins have been associated with various cellular activities, including receptor activity, cell-cell binding, integrin binding and/or signaling, or channel activity, e.g., Ca2+ channel activity (see, e.g., Bubien et al., J Cell Bio 121(5):1121 (1993)).
Glycophorin A (CD235a) is a 131-amino acid asialoglycoprotein that spans the membrane of the erythrocyte once. CD235a has an extracellular peptide portion that is antigenic.
The National Institutes of Health (USA) has conducted a Phase II/III study evaluating the effect of IL-2 on preservation of the CD4 Th-lymphocytes after interruption of anti-retroviral treatment (AVT) of infected patients with CD4 Th-lymphocyte counts greater than 500 cells/mm3 who had received AVT (NCTOO071890 Identifier). Th1s study examined whether interleukin-2 (IL-2) given before the interruption of anti-retroviral (ART) treatment could significantly extend the period of time that a patient is temporarily not taking ART treatment and also preserve CD4 counts above 350 cells per microliter. There was an evaluation of the toxicity, or extremely harmful effects, of ART, and the effect on quality of life. The use of ART medications has greatly improved the condition and mortality of HIV-infected patients. But when used long term, those medications have been associated with great toxicities and medication fatigue. As a result, patients may not adhere to ART use, and resistance to viruses may grow. The CD4 molecule is on the surface of Th-lymphocytes, or It serves as the primary receptor for HIV-1 and HIV-2, allowing the virus to gain entry into its host. The CD4 count increases immediately in response to ART, giving an estimate of the state of a patient's immune system. Thus, it is a strong marker of the immediate risk of an opportunistic infection, one that takes advantage of a person's weakened immune system. IL-2 is a molecule naturally produced by activated T cells. In patients with HIV, IL-2 treatment can increase CD4 counts but the clinical importance of this increase was not clear. This study compared the decline in Th CD4 count, when ARV was interrupted. This type of clinical study would be greatly aided were there a simple means of isolating Th lymphocytes from patient blood quickly and easily.
Rapid access to “clean” Th1 and Th2 lymphocytes would also be an asset in: studies of the reduction of Th1 lymphocytes in peripheral blood in: Grave's Disease and Type 1 Diabetes (Matsuura, A Y et al., Endocrin. J. May 23, 2006); immunotherapeutics (Wang, C Y et al., Vaccine; 23:49 (2005)); tracking the severity of human asthma or assessing the effectiveness of immunomodulating drugs used to treat asthma and other autoimmune diseases; and, other Th1/Th2-balance related diseases, e.g., where allergy skin testing by RAST is not possible; and, other antigen-related diseases.
These goals have been achieved by the development of a novel and useful method of isolating, separately, Th1 and Th2 lymphocytes from human peripheral blood. This method is described below.